Poster: Membrane Transport

Abs # 1220: Development of a novel high-throughput assay to identify and characterize inwardly conducting Na channels and use to demonstrate Na conductance of a plant cyclic nucleotide gated channel

Presenter:	Berkowitz, Gerald A, gerald.berkowitz@uconn.edu
Authors	Berkowitz, Gerald A (A)   Gaxiola, Roberto A. (A)
Affiliations:	(A): University of Connecticut

Identification of specific plant genes associated with ion transport has been facilitated by use of appropriate yeast mutants. However, at present the only such screen for inwardly-conducting Na-permeable transporters or channels using yeast is hypersensitivity of mutants lacking Na detoxification systems. For example, the ena1 (Na efflux ATPase) yeast mutant showed Na hypersensitivity (i.e. reduced growth in presence of Na) upon expression of the plant Na transporters AtHKT1 and LCT1. Hygromycin B (Hyg) is a toxic cation that enters the yeast cytosol in response to cell membrane potential. Influx of cations (K or Na) depolarizes the yeast cell membrane; reducing Hyg uptake. The nhx1 (vacuolar cation/H antiporter) mutant is hypersensitive to Hyg presumably due to reduced capacity for vacuolar sequestration of Hyg. Hypersensitivity of nhx1 to Hyg (evaluated by relative growth in liquid culture) can be reversed by addition of K (and Na at higher concentrations) to the growth medium. Expression of the plant cyclic nucleotide gated nonselective cation channel AtCNGC1 in nhx1 yeast complemented hypersensitivity to Hyg when low [Na] and a lipophilic analog of cAMP were added to the growth medium. Voltage clamp analysis of AtCNGC1 expressed in HEK cells confirms Na permeation. AtCNGC1 and homologs are the first cloned plant channels shown to conduct Na and may contribute to Na uptake into plants. Our work also demonstrates the efficacy of this yeast mutant screen as a high throughput system to functionally characterize cloned inward Na transporters. The development of such a positive selection screen could facilitate structure/function studies of Na channels and provide a high throughput assay for discovery of drugs that modulate Na channels. Supported by NSF award 0090675.